Why certain patients respond to chemotherapy and others do not remain an unsolved problem in medical oncology. Through the years, we have achieved a more sophisticated understanding of the molecular determinants of tumor responsiveness. One of the most exciting advances was the discovery of P-glycoprotein (P-gp), the MDR1 gene product, which can impart multi-drug resistance (MDR) by virtue of its ability to transport certain molecules out of the cell. By identifying and then developing clinically safe modulators of P-gp, patients with highly resistant tumors are now being offered a chance at greater responsiveness, and hopefully, a longer life. Let, with few exceptions, the use of MDR modulators in the clinic has not been highly successful. In this proposal, we continue to pursue our goal of understanding the mechanisms of drug resistance and developing the means to overcome them. We previously defined the structure-activity relationships for several classes of MDR modulators, synthesized irreversible P-gp antagonists, and developed animal models for drug testing. We recently made the disturbing observation that treatment of animals bearing P-gp (+) tumors with P-gp substrates (chemotherapeutics or modulators) decreased overall survival and increases metastases. Preliminary studies suggest that this may be due to the ability of P-gp substrates to selectively affect cell motility and invasiveness in P-gp (+) cell lines. An alternative approach to the problem of MDR is determine the mechanism(s) by which cells acquire the MDR phenotype and how this can be prevented or overcome. We found that activation of phospholipase C by growth factors or physical stimuli set off a signal transduction cascade that activates MDR1 transcription. Since these results raise the possibility that cells can acquire the MDR phenotype through the MAP kinase pathways, therefore, by understanding this pathway and developing inhibitors, it may be possible to prevent emergence of P-gp(+) cells in the tumor population. Finally, our studies on the control of MDR1 gene expression found that mutant p53 produced resistance to several P-gp substrates including vinca alkaloids and anthracyclines, but produced collateral sensitivity to taxanes. This was not due to changes in P-gp but rather due to the de- repression of a microtubule associated protein, MAP4. MAP44 polymerizes microtubules, which leads to increased cellular binding of taxanes and decreased binding of vinca alkaloids. We also found a correlation between expression of mutant p53 in human prostate cancer specimens and expression of the multi-drug resistance protein (mrp). In the next grant period we will focus on several of these observations, which should lead to a cleared understanding of the mechanisms underlying sensitivity to anti-cancer drugs and the implications of trying to overcome them.